Naslov
Key Residues for Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution

Autori
Tang, Lixia ; Zhu, Xuechen ; Zheng, Huayu ; Jiang, Rongxiang ; Majerić Elenkov, Maja

Izvornik
Applied and environmental microbiology (0099-2240) 78 (2012), 8; 2631-2637

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
halohydrin dehalogenase; enantioselectivity; semi-rational design; kinetic resolution

Sažetak
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of (R)-enantiomers of epoxides and β- substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low (S)-enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) lied located in the two neighboring active-site loops of halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA with a low S-enantioselectivity toward model substrate 2-chloro-1-phenylethanol were proposed to be the key residues for controlling enantioselectivity. They were and subjected to saturation mutagenesis aimed at evolving a (S)-selective enzyme. This lead to the selection of two outstanding mutants (V136Y/L141G and N178A). The double mutant displayed an inverted enantioselectivity (from ES = 2 1.5 to ER = 138) toward 2-chloro-1- phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (ES > 200) and a 5-fold enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of the non-favored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that the a semi-rational design strategy has a great potential for rapid creation of novel industrial biocatalysts.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biotehnologija

POVEZANOST RADA